Pharmaceutical compositions containing parthenium integrifolium or parts thereof or an extract or component thereof, the use of such plant material for preparing certain medicines, and a method of preparing an extract of parthenium integrifolium

ABSTRACT

The use of  Parthenium integrifolium  (PI) or its parts, extract or components, for enhancement of the TH2 pathway of the immune system, enhancement of levels of interleukin-4 and -10, and selective suppression of cyclooxygenase-2. Also claimed are: (1) composition containing PI; and (2) extracts of PI and their preparation. PI is particularly useful for alleviation of pain (e.g. migraine or headache), and treatment or prevention of inflammatory or autoimmune disorders.

This is a continuation of International Application No. PCT/DK97/00457,filed Oct. 20. 1997, which claims priority of U.S. Provisionalapplication Ser. No. 60/031,395, filed Nov. 19, 1996.

FIELD OF THE INVENTION

The present invention relates to the plant Parthenium integrifolium andmore specifically to pharmaceutical compositions derived from it as wellas the use of Parthenium integrifolium or parts thereof or an extract orcomponent thereof for the preparation of medicines for the alleviationof pain or for the treatment or prevention of inflammatory or autoimmunedisorders. The invention also relates to a method of preparing anextract of Parthenium integrifolium and to the extracts prepared by themethod.

BACKGROUND OF THE INVENTION

Parthenium integrifolium (L.) (family Asteraceae), also commonly knownas Missouri snake root, grows wild in woodland and prairies of NorthAmerica. The herb is 30-130 cm high with numerous white flowerheadsforming a flat inflorescence up to 25 cm wide. The root is comprised ofa short, conical or bulbshaped headstem that has a diameter of up to 4cm and elongated secondary, twisted branches leading from the headstem.

A number of chemicals have been identified as major components ofParthenium integrifolium extracts. One group are the sesquiterpenelactones represented by tetraneurin E and tetraneurin C. Another groupare the sesquiterpene esters represented by echinadiol cinnamate, epoxyechinadiol cinnamate, echinaxanthol cinnamate and dihydroxynardolcinnamate. Yet another group are the flavonoids represented byquercetagetin methyl ethers and their O-glycosides. Anothercharacteristic component of Parthenium integrifolium is pyromeconicacid. Other chemicals present in the plant are coumarins and diversephenolic glycosides.

The German patent application, publication no. 36 38 715 A1 describesthe above mentioned sesquiterpene esters derived from Partheniumintegrifolium. According to the experimental section of thatapplication, immunological activity tests of the sesquiterpene estersshowed that they enhanced granulocyte phagocytosis in vitro up to 30%.This effect is to be considered a pro-inflammatory action related to thenon-specific part of the immune system (the reticuloendothelialphagocytic system).

At present the nonsteroidal antiinflammatory drugs (NSAIDS) are the mostcommonly applied therapeutic agents for the treatment of conditionsassociated with inflammation and pain. The NSAIDs exert their action byinhibiting the prostaglandin-generating enzyme cyclooxygenase (COX).There are two biochemical subtypes of cyclooxygenase denominated COX-1and COX-2. COX-1 is constitutively expressed in most cells and isresponsible for the formation of prostaglandins which mediate importantbasic physiological functions, e.g. providing an intact mucosa in theventricle. COX-2 is not normally present, but may be induced by certainserum factors, cytokines and growth factors and responsible for theformation of inflammatory prostaglandins which mediate many symptoms ofinflammation. The NSAIDs are generally non-selective, meaning that theyinhibit both COX-2 and COX-1 resulting in an antiinflammatory and painrelieving effect due to the inhibition of COX-2 and a number of sideeffects due to the inhibition of COX-l, of which gastric ulceration isone of the most important.

Autoimmune disorders like multiple sclerosis, morbus Crohn, rheumatoidarthritis, diabetes mellitus, etc. are associated with an overactivationof the inflammatory arm of the immune system (T_(H)1 pathway) leading towell known symptoms and serious tissue destruction. The most wellestablished treatment for these disorders is the management ofcorticosteroids which exert their action by non-selectively inhibitingthe function and proliferation of different types of immune cells.Unfortunately the corticosteroids are associated with a number ofserious side effects e.g. immuno-suppression and osteoporosis.

SUMMARY OF THE INVENTION

I have found that Parthenium integrifolium or parts thereof or anextract or component thereof exert the following pharmacologicalactions: Enhancement of the T_(H)2 pathway of the immune system,enhancement of the levels of interleukin-4 and interleukin-10,suppression of cyclooxygenase-2 (COX-2), reduction of chronic and acutepain, and reduction of inflammation. Compared to the NSAIDs Partheniumintegrifolium or parts thereof or an extract or component thereof havethe advantage that they are not associated with gastrointestinal andrenal side effects. Further, by enhancing the formation of interleukin-4and interleukin-10 they have a down regulating effect on the T_(H)1pathway of the immune system without exerting the serious side effectscharacteristic of the corticosteroids. Due to these effects Partheniumintegrifolium or parts thereof or an extract or component thereof can beemployed for the following therapeutic applications:

Alleviation of pain.

Treatment or prevention of inflammatory or autoimmune disorders.

Accordingly the present invention provides a pharmaceutical compositioncontaining Parthenium integrifolium or parts thereof or an extract orcomponent thereof and a pharmaceutically acceptable carrier.

More specifically the present invention provides the use of Partheniumintegrifolium or parts thereof or an extract or component thereof forpreparing a medicine for the enhancement of the T_(H)2 pathway of theimmune system, for the enhancement of the levels of interleukin-4 andinterleukin-10, and for the selective suppression of COX-2.

Thus, according to the invention Parthenium integrifolium or partsthereof or an extract or component thereof can be used in a method forthe alleviation of pain in an individual, which comprises administeringsuch plant material or a pharmaceutical composition containing it tosaid individual; and the invention comprises the use of Partheniumintegrifolium or parts thereof or an extract or component thereof forpreparing a medicine for the alleviation of pain.

Also, according to the invention Parthenium integrifolium or partsthereof or an extract or component thereof can be used in a method forthe treatment or prevention of an inflammatory or autoimmune disorder inan individual, which comprises administering such plant material or apharmaceutical composition containing it to said individual; and theinvention comprises the use of Parthenium integrifolium or parts thereofor an extract or component thereof for preparing a medicine for thetreatment or prevention of inflammatory or autoimmune disorders.

Further, the invention provides a method of preparing an extract ofParthenium integrifolium, which comprises extracting said plant or partsthereof, preferably the root, with an extraction agent comprising anorganic solvent or a mixture thereof with water and subsequently, ifnecessary, removing the extraction agent to obtain an extract suitablefor utilisation.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly it has been found that Parthenium integrifolium or partsthereof or an extract or component thereof exert pharmacological actionsrelevant to the therapeutic treatment of conditions associated withpain, inflammation and autoimmunity.

More specifically Parthenium integrifolium or parts thereof or anextract or component thereof provide the following pharmacologicaleffects upon administration to the living organism:

Enhancement of the T_(H)2 pathway of the immune system.

Enhancement of the levels of interleukin-4 and interleukin-10.

Suppression of COX-2 without affecting COX-1.

Reduction of pain.

Reduction of inflammation.

These actions provide part of the rationale for the followingtherapeutic applications of Parthenium integrifolium or parts thereof orextracts or components thereof:

A method for the treatment of any condition associated with pain orinflammation characterised by the administration of Partheniumintegrifolium or parts thereof or an extract or component thereof. Theapplicant puts forward the hypothesis that the antiinflammatory and painrelieving action is due to enhanced levels of interleukin-4 and -10which on one hand down regulate the inflammatory part of the immunesystem (T_(H)1 pathway) and on the other hand reduce the expression ofCOX-2 leading to a lower level of inflammatory prostaglandins. Thismechanism of action holds the advantage compared to conventionalnonsteroidal antiinflammatory drugs (NSAIDs) that COX-1 is not inhibitedwhereby gastric ulceration and renal side effects are avoided. In apreliminary clinical observation a patient with pain and inflammationdue to osteoarthritis in the lower back experienced a significantimprovement after treatment with 250 mg a day of the extract ofParthenium integrifolium described in example 1. The patient wassubjected to an oral dose of 125 mg of the extract twice a day,administered in a hard gelatine capsule. The improvement which consistedin a total elimination of pain and increased mobility in the affectedjoints was apparent on day 2 of treatment and continued for the entireperiod of treatment during 3 weeks. After termination of the treatmentthe pain returned after two days. The clinical improvement is attributedto the above mentioned effects of Parthenium integrifolium. In theclinical pilot study described in example 4 patients (n=16) sufferingfrom migraine headache were subjected to a daily dose of an extract ofParthenium integrifolium. The study schedule consisted of four periodsof four weeks. The first period served as a baseline, where no treatmentwas employed. In the second period a daily dose of 200 mg Partheniumintegrifolium extract was employed. In the third period a daily dose of100 mg Parthenium integrifolium extract was employed. In the fourthperiod a daily dose of 200 mg Parthenium integrifolium extract wasemployed. During the four periods all incidences of migraine headachewere recorded. The incidence of migraine headache was reduced in all ofthe treatment periods as compared to the baseline period. In the lasttreatment period the incidence of migraine headache was reduced by 57%,which was statistically significant (p<0,05; Wilcoxon). The observedclinical improvement is attributed to the above mentioned effects ofParthenium integrifolium.

A method for the treatment of inflammatory and autoimmune disorderscharacterised by the administration of Parthenium integrifolium or partsthereof or an extract or component thereof. The applicant puts forwardthe hypothesis that the therapeutic action is due to enhanced levels ofinterleukin-4 and -10 which down regulate the T_(H)1 pathway of theimmune system which plays a significant role in the pathogenesis ofinflammatory and autoimmune disorders. Furthermore interleukin-4 and -10down regulate the expression of COX-2 leading to a decreased formationof inflammatory prostaglandins and a reduction of symptoms of acuteinflammation. The therapeutic action may be relevant to all knownautoimmune or inflammatory diseases and the following examples are notlimiting with respect to this: Autoimmune hepatitis, Primary biliarycirrhosis, Primary sclerosing cholangitis, Autoimmune hemolytic anemias,Grave s disease, Myastenia gravis, Type 1 Diabetes Mellitus,Inflammatory myopathies, Multiple sclerosis, Hashimoto's thyreoiditis,Autoimmune adrenalitis, Crohn Disease, Ulcerative Colitis,Glomerulonephritis, Progressive Systemic Sclerosis (Scleroderma),Sögren's Disease, Lupus Erythematosus, Primary vasculitis, ReumatoidArthritis, Juvenile Arthritis, Mixed Connective Tissue Disease, AtopicDermatitis, Psoriasis, Pemfigus, Pemfigoid, Dermatitis Herpetiformis,etc.

The preferred embodiment of the invention is an extract of Partheniumintegrifolium. Extracts according to the invention can i.a. be obtainedby extraction or by steam or vacuum distillation of fresh or driedParthenium integrifolium or parts thereof, preferably the root.Extraction may be performed with a number of different organic solvents,preferably water miscible solvents, and mixtures thereof with water. Theextraction can be performed hot or cold by the employment of anyextraction technology e.g. maceration, percolation or supercriticalextraction.

The preferred extraction solvents are acetone, methyl ethyl ketone,ethyl acetate, lower alkanols having 1 to 4 carbon atoms and mixturesthereof with water. The preferred extraction temperature is close to theboiling point of the employed solvent due to extraction efficacy, butlower temperatures are also applicable making necessary a longer periodof extraction.

By changing the composition of the applied solvent the extraction can bemade more selective for certain constituents of Parthenium integrifoliumthus enhancing or reducing their content in the finished extract. Forexample the content of phenolic glycosides can be increased by employinga more hydrophilic solvent while the content of sesquiterpenes in thefinished product can be enhanced by employing a more lipophilic solvent.

After the primary extraction process a second step of processing, suchas liquid-liquid extraction, column chromatography, steam distillationor vacuum distillation, can be employed to remove or to concentrate andpossibly isolate any constituent of the extract. Hereby any constituentof Parthenium integrifolium can be avoided or concentrated in thefinished extract, e.g. pyromeconic acid, sesquiterpene lactones liketetraneurin E and tetraneurin C, sesquiterpene esters like echinadiolcinnamate, epoxy echinadiol cinnamate, echinaxanthol cinnamate anddihydroxynardol cinnamate, flavonoids like quercetagetin methyl ethersand their O-glycosides, coumarins or various phenolic glycosides. Thusthe content of any component of Parthenium integrifolium can bestandardised in the finished extract for the purpose of manufacturing apharmaceutical composition.

According to the invention Parthenium integrifolium or parts thereof oran extract or component thereof can be combined with any other activeingredient or plant extract to potentiate the therapeutic action.Consequently, we propose to combine Parthenium integrifolium or partsthereof or extracts or components thereof with eicosapentaenoic acidfrom fish oils or γ-linolenic acid for the treatment of inflammatory orautoimmune disorders. As a parallel, we propose to combine Partheniumintegrifolium or parts thereof or extracts or components thereof withZingiber officinale or parts thereof or extracts or components thereoffor the treatment of pain and inflammation.

Furthermore it is obvious that in the use according to the invention forpreparing medicines Parthenium integrifolium or parts thereof or anextract or component thereof may be mixed with additives such assurfactants, solvents, thickeners, stabilisers, preservatives,antioxidants, flavour etc. to obtain a desirable product formulation.Similarly, the pharmaceutical compositions according to the inventionmay further contain such additives. There are no limitations to thedosage form of the formulation, but tablets, gelatine capsules, fluidsor granulates are envisaged. Optionally, the composition may alsocontain surfactants such as bile salts or polyoxyethylene-sorbitan-fattyacid esters for improving dispersibility of the composition in thedigestive fluids leading to improved bioavailability or for obtainingthe final dosage form of the composition.

EXAMPLES Example 1

An extract of Parthenium integrifolium according to the invention wasprepared as follows:

100 g dried root of Parthenium integrifolium was extracted with 1500 mlof boiling 90% ethanol for 4 hours. Thereafter the extract was filteredand evaporated to dryness under vacuum. Thus 22 g of an amber-colouredcrystalline extract was obtained suitable for the manufacture of tabletsor hard gelatine capsules.

Example 2

An extract of Parthenium integrifolium according to the invention wasprepared as follows:

100 g dried root of Parthenium integrifolium was extracted with 1500 mlof boiling 96% ethanol for 5 hours. Thereafter the extract was filteredand evaporated to dryness under vacuum. 15 g of an amber-colouredcrystalline extract was obtained. This Parthenium integrifolium extractwas diluted in 30 ml 80% ethanol, and to this was added 15 g ofacetylated monoglyceride and 5 g of polyoxyethylene-sorbitan-monooleate(Tween 80). Thus a liquid extract was obtained suitable for themanufacture of soft gelatine capsules.

Example 3

Materials and Methods

Mice

BALB/c mice, 5-6 weeks old were purchased from Gl. Bomholtgaard, Ry,Denmark. Unless otherwise specified they were fed standard food pelletsand water ad libitum.

Test Compound (Extract of Parthenium integrifolium)

An extract of Parthenium integrifolium according to the invention wasprepared as follows:

100 g dried root of Parthenium integrifolium was extracted with 1500 mlof boiling 80% ethanol for 3 hours. Thereafter the extract was filteredand evaporated to dryness under vacuum. Thus 24 g of an amber-colouredcrystalline extract was obtained. This Parthenium integrifolium extractis abbreviated PI in the rest of this example.

Feeding Regime

Mice were fed crushed ordinary mouse pellets and water ad libitum.Crushed mouse pellets were exposed to PI or placebo: An ethanolicsolution of PI was prepared and mixed with crushed standard mousepellet. After drying the content of PI was 6 mg/kg mouse pellet. A mousewas estimated to consume 5 g standard mouse pellet a day resulting in adaily intake of 0,30 mg PI corresponding to a daily dosage of 10 mg/kgat an average body weight of 30 g/mouse. The control diet was preparedby substituting the PI ethanolic solution with pure ethanol in the abovementioned procedure.

Reagents

Sheep red blood cells (SRBC) were purchased from Statens Seruminstitut,Copenhagen Denmark. Antibodies for Elisa assay were purchased fromPharmingen, San Diego, Calif., U.S.A (see below). The SRBC were washedthree times in physiological saline prior to use.

SRBC plaque forming cells (SRBC-PFC)

Four days after intravenous injection of 0.2 ml 10% SRBC inphysiological saline, the mice were sacrificed and their spleens removedand homogenised by pressing the organs gently through a metal net. Thecells were counted and mixed with SCRB and rabbit complement, and thecell mixture transferred to a reaction chamber (Cunningham andStzenberg) for quantitation of SRBC-PFC. Four individual assay chamberswere counted per splenocyte preparation. The numbers of PFC werecalculated as numbers per 10⁶ splenocytes.

Anti-SRBC Antibody Titres

Mice were injected intraperitoneally with 10% SRBC in physiologicalsaline in a volume of 0.5 ml. One hundred ml blood were collected fromthe retroorbital venous plexus at day 3, 6 and 9 post immunisation andtransferred to vials containing 100 ml saline with 2 units of heparin.Plasma (50% dilution) was recovered by centrifugation and serialdilutions performed in round bottom microtitre plates (Nunc,Roskilde,Denmark). SRBC 0.1% and freshly diluted rabbit complement (Glapco,Jylland, Denmark) was added and the plates were incubated at 37° C. for2 hours. Hemolysin titers of individual plasma samples were read by eyeas the highest plasma dilution giving total lysis of the added SRBC.

Mixed Lymphocyte Culture (MLC)

Spleen cells obtained from pools of 5 spleens were cultured in volumesof 10 ml (2×10⁶) per ml in 25 ml T flasks and stimulated with irradiatedC57BL6 splenocytes (10⁶/ml). One ml culture supernatants were removed atday 3 and 4 of culture for cytokine determination.

Cytokines Secreted by MLC Responder Cells

Elisa assays for IL-2, IL-4 and IL-10 were set up using reagents fromPharmingen: IL-2 standard 1921IU, IL-4 standard1923IW and IL-10 standard1228IV. Antibodies: anti-IL-2 18161D, anti-IL4 18031D and anti-IL-1018141D (capture Abs), anti-IL-2 18172D, anti-IL-4 18042D and anti-IL-1018152D (biotinylated detection Abs). Dose-response cytokine standardcurves were generated. The three different cytokines secreted by the MLCresponder cells at day 3 and 4 of culture were determined from replicatedilutions of the MLC culture supernatants. The linear part of thestandard cytokine curves were used to determine the amounts of theindividual cytokines.

Statistics

Wilcoxon rank sum test for paired differences and Fischers exact testwere used for comparing anti-SRBC antibody titers and SRBC-PFC numbersrespectively in PI and placebo fed mice.

Results

Both PI and placebo fed animals tolerated crushed mouse pellets well andno weight loss was registered in any of the experimental or placebotreated mice.

SRBC-PFC

The individual numbers of SRBC-PFC per 10⁶ splenocytes of two groups of21 mice were derived from three separate experiments. The mice were fedPI or placebo for ten consecutive days and immunised with SRBC at day 6and killed at day 10 of the feeding regime. The mean numbers of SRBC-PFCper 10⁶ splenocytes in the two groups were 400 and 220, respectively.However, these numbers were not statistically different (p>0.05,Wilcoxon). However, some differences among the two groups of mice wereencountered. Thus, six of the 21 mice in the PI fed group and only 1 of21 mice in the placebo group produced more than 500 PFC per 10⁶splenocytes, this difference between the two groups being significant(p<0.02, Ficher's exact test). Moreover, when the ten mice producing thehighest number of SRBC-PFC in each of the two groups were compared thedifference between these two high responding “subgroups” was significant(P<0.05,Wilcoxon).

Anti-SRBC hemolysins

The results from the SRBC-PFC study prompted us to examine the level ofanti-SRBC antibody titers from individual mice fed for 14 days with PIor placebo respectively. Table 1 shows the results. The PI fed group ofmice showed significantly higher (p<0.005) anti-SRBC titer values at day6 after immunisation as compared with the placebo fed group of mice.

TABLE 1 Anti-SRBC hemolysin titres in mice fed EPC-10 or placebo for 12days. Mice were immunized at day 3 and antibody titers determined 3, 6and 9 days after immunization. Days post MICE FED WITH: immunization PIPlacebo 3 7 (0-18)* 18 (0-36) 6 691# (576-1152) 245 (72-288) 9 202(144-288) 115 (72-144) *Numbers in parentheses represent the range oftiters in five individual mice #Significantly different from the placebofed group, p<0.005 (Wilcoxon)

Cytokines

Supernatants from the MLC cultures described above were assayed forcytokine content at day 3 and 4 of culture. The amounts of IL-2, IL-4and IL-10, respectively, were measured in the MLC supernatants by asensitive ELISA technique. As shown in Table 2, at day 3 of culture, theMLC supernatants from PI fed mice contained two times more IL-2 andIL-10 and seven times more IL-4. At day 4 of culture supernatants fromthe PI fed mice contained three times more IL-10 and twice as much IL-4compared with MLC supernatants of placebo fed animals.

TABLE 2 Amounts* of IL-2, IL-4 and IL-10 secreted by allo-stimula- tedsplenocytes obtained from PI or placebo fed mice. PI Placebo Days ng/mlpg/ml ng/ml pg/ml of MLC IL-2 IL-10 IL-4 IL-2 IL-10 IL-4 3 13.5 2.6 1916.2 1.3 26 4  0.9 2.9  54 0.7 1.0 27 *Amounts of cytokine per ml culturesupernatant produced by MLC responder cells from a pool of five spleens,numbers represent the means of two replicate cytokine measurements.

Example 4

Study Object

An extract of Parthenium integrifolium according to the invention wasprepared as follows:

1000 g dried root of Parthenium integrifolium was extracted with 10 000ml of boiling 90% ethanol for 4 hours. Thereafter the extract wasfiltered and evaporated to dryness under vacuum. Thus 216 g of anamber-coloured crystalline extract was obtained. Tablets containing 50mg of the extract were prepared.

Study Summary

Background

The object of the study was to evaluate the prophylactic effect of theextract of Parthenium integrifolium on migraine headache in an openclinical trial.

Methods

16 migraine sufferers with a history of at least four incidences ofmigraine headache a month during the last six months were included inthe study.

The study schedule consisted of four periods of four weeks. The firstperiod served as a baseline, where no treatment was employed. In thesecond period a daily dosage of 4 tablets was employed. In the thirdperiod a daily dosage of 2 tablets was employed. In the fourth period adaily dosage of 4 tablets was employed. During the four periods allincidences of migraine headache were recorded.

Statistics

Wilcoxon rank sum test for paired differences was used for comparing theincidence of migraine headache in each of the three treatment periodswith the incidence in the baseline period.

Findings

The mean incidence of migraine headache was 5,75 in period 1 (baseline).The mean incidence of migraine head-35 ache was 30% lower in period 2,43% lower in period 3 and 57% lower in period 4 as compared to baseline.Period 4 was significantly different from baseline (p<0.05; Wilcoxon).

Interpretation

In this study the tested extract of Parthenium integrifolium wasconcluded to be a powerful remedy in the prophylaxis of migraineheadache.

What is claimed is:
 1. An extract of Parthenium integrifolium, obtained by extraction of the plant or parts thereof with a water miscible organic solvent or a mixture thereof with water.
 2. An extract according to claim 1, obtained by extraction with a water miscible organic solvent selected from the group consisting of acetone, methyl ethyl ketone, ethyl acetate or lower alkanols having 1 to 4 carbon atoms.
 3. A pharmaceutical composition comprising an extract of Parthenium integrifolium according to claim 1 and a pharmaceutically acceptable carrier.
 4. A pharmaceutical composition comprising the Parthenium integrifolium extract of claim 1 together with γ-linolenic acid or eicosapentaenoic acid and a pharmaceutically acceptable carrier.
 5. A pharmaceutical composition comprising the Parthenium integrifolium extract of claim 1 together with Zingiber officinale or parts thereof or an extract or component thereof and a pharmaceutically acceptable carrier.
 6. A pharmaceutical composition comprising an extract of Parthenium integrifolium according to claim 2 and a pharmaceutically acceptable carrier. 